Identification and characterization of a Masculinizer homologue in the diamondback moth,Plutella xylostella

Recently, a novel sex‐determination system was identified in the silkworm (Bombyx mori) in which a piwi‐interacting RNA (piRNA) encoded on the female‐specific W chromosome silences a Z‐linked gene (Masculinizer) that would otherwise initiate male sex‐determination and dosage compensation. Masculinizer provides various opportunities for developing improved genetic pest management tools. A pest lepidopteran in which a genetic pest management system has been developed, but which would benefit greatly from such improved designs, is the diamondback moth, Plutella xylostella. However, Masculinizer has not yet been identified in this species. Here, focusing on the previously described ‘masculinizing’ domain of B. mori Masculinizer, we identify P. xylostella Masculinizer (PxyMasc). We show that PxyMasc is Z‐linked, regulates sex‐specific alternative splicing of doublesex and is necessary for male survival. Similar results in B. mori suggest this survival effect is possibly through failure to initiate male dosage compensation. The highly conserved function and location of this gene between these two distantly related lepidopterans suggests a deep role for Masculinizer in the sex‐determination systems of the Lepidoptera.     

Identification and characterization of doublesex in Bemisia tabaci

Bemisia tabaci (Gennadius) is an important agricultural pest with a worldwide distribution. Although B. tabaci is known to have a unique haplodiploid reproductive strategy, its sex determination mechanism is largely unknown. In this study, we cloned the full‐length sequence of B. tabaci doublesex (Btdsx) and found that Btdsx has 28 splicing isoforms. We found two new splicing isoforms of transformer 2 (Bttra2), which encode two proteins. We also confirmed that both genes lack sex‐specific splicing isoforms. Real‐time quantitative PCR analysis showed that the expression of Btdsx and Bttra2 is higher in males than in females. RNA interference of Bttra2 affected the expression of Btdsx and vice versa. Furthermore, silencing of Bttra2 or Btdsx caused malformation of the male genitalia (anal style). It did not affect the female phenotype, but reduced the expression of vitellogenin gene in females. These results indicate that Btdsx is associated with sex determination in B. tabaci and that Btdsx and Bttra2 affect each other and are important for male genitalia formation. In addition to increasing our understanding of the roles of dsx and tra2 in the sex determination of B. tabaci, the results will be useful for studies of sex determination in other haplodiploid species.     

Maternal provision of non‐sex‐specific transformer messenger RNA in sex determination of the wasp Asobara tabida

In many insect species maternal provision of sex‐specifically spliced messenger RNA (mRNA) of sex determination genes is an essential component of the sex determination mechanism. In haplodiploid Hymenoptera, maternal provision in combination with genomic imprinting has been shown for the parasitoid Nasonia vitripennis, known as maternal effect genomic imprinting sex determination (MEGISD). Here, we characterize the sex determination cascade of Asobara tabida, another hymenopteran parasitoid. We show the presence of the conserved sex determination genes doublesex (dsx), transformer (tra) and transformer‐2 (tra2) orthologues in As. tabida. Of these, At‐dsx and At‐tra are sex‐specifically spliced, indicating a conserved function in sex determination. At‐tra and At‐tra2 mRNA is maternally provided to embryos but, in contrast to most studied insects, As. tabida females transmit a non‐sex‐specific splice form of At‐tra mRNA to the eggs. In this respect, As. tabida sex determination differs from the MEGISD mechanism. How the paternal genome can induce female development in the absence of maternal provision of sex‐specifically spliced mRNA remains an open question. Our study reports a hitherto unknown variant of maternal effect sex determination and accentuates the diversity of insect sex determination mechanisms.     

Fine scale mapping in the sex locus region of the honey bee (Apis mellifera)

Isolating an unknown gene with fine-scale mapping is possible in a "non-model" organism. Sex determination in honey bees consists of a single locus (sex locus) with several complementary alleles. Diploid females are heterozygous at the sex locus, whereas haploid males arise from unfertilized eggs and are hemizygous. The construction of specific inbred crosses facilitates fine scale mapping in the sex locus region of the honey bee. The high recombination rate in the honey bee reduces the physical distance between markers compared with model organisms and facilitates a novel gene isolation strategy based on step-wise creation of new markers within small physical distances. We show that distances less than 25 kb can be efficiently mapped with a mapping population of only 1000 individuals. The procedure described here will accelerate the mapping, analysis and isolation of honey bee genes.     

Cloning and functional expression of a human orthologue of rat vanilloid receptor-1

Capsaicin, resiniferatoxin, protons or heat have been shown to activate an ion channel, termed the rat vanilloid receptor-1 (rVR1), originally isolated by expression cloning for a capsaicin sensitive phenotype. Here we describe the cloning of a human vanilloid receptor-1 (hVR1) cDNA containing a 2517 bp open reading frame that encodes a protein with 92% homology to the rat vanilloid receptor-1. Oocytes or mammalian cells expressing this cDNA respond to capsaicin, pH and temperature by generating inward membrane currents. Mammalian cells transfected with human VR1 respond to capsaicin with an increase in intracellular calcium. The human VR1 has a chromosomal location of 17p13 and is expressed in human dorsal root ganglia and also at low levels throughout a wide range of CNS and peripheral tissues. Together the sequence homology, similar expression profile and functional properties confirm that the cloned cDNA represents the human orthologue of rat VR1.     

Identification,characterization and functional analysis of a chitin synthase gene in the brown citrus aphid,Toxoptera citricida (Hemiptera,Aphididae)

Chitin synthase (CHS) is a crucial enzyme involved in the final step of the insect chitin biosynthetic pathway. In this study, we cloned the full‐length cDNA sequence of a chitin synthase gene (TCiCHS) from the brown citrus aphid, Toxoptera citricida, an important citrus pest and the main vector of citrus tristeza virus worldwide. TCiCHS was expressed during the entire lifecycle and in all insect tissues examined. Expression was highest in first–second‐instar nymphs, nymph–adult transitions and in the abdomen (6.7‐fold higher than head). Embryos had a higher expression level than the integument. Fourth‐instar nymphs were exposed to 5 and 500 mg/l concentrations of the chitin synthesis inhibitor diflubenzuron (DFB) for 48 h and had the highest mortality at the 500 mg/l concentration. The mRNA expression levels of TCiCHS were significantly enhanced upon the exposure of nymphs to both low and high DFB concentrations. Silencing of TCiCHS occurred through plant‐mediated double‐stranded RNA (dsRNA) feeding. Most dsRNA‐fed nymphs were unable to moult to the next stage, and the expression of TCiCHS decreased 48% compared with controls. These results demonstrate that TCiCHS plays an important role in nymph to adult development, is possibly help identify molecular targets for To. citricida control.     

A lepidopteran orthologue of reaper reveals functional conservation and evolution of IAP antagonists

Genetic studies in Drosophila melanogaster have revealed that inhibitor of apoptosis (IAP) proteins and IAP antagonists such as reaper play a pivotal role in controlling cell death in insects. Interestingly, although the sequences and structures of IAPs are highly conserved, the sequence of IAP antagonists diverged very rapidly during evolution, making their identification difficult. Using a customized bioinformatics approach, we identified an IAP antagonist, IAP-binding motif 1 ( Ibm1 ), from the genome of the silkworm Bombyx mori . This is the first reaper/grim orthologue identified in a nondipteran insect. Previous analysis indicated that both Reaper and Grim induce cell death through their N-terminal IBM as well as the Grim_helix3 (GH3) domain. Functional studies indicated that Ibm1 binds to an IAP protein from B. mori , BmIAP1, and induces apoptosis in insect cells via the IAP-binding motif, a seven amino acid sequence that is highly conserved in all IAP antagonists. Interestingly, Ibm1 also contains a region that is a statistically significant match to the GH3 domain. Mutational analysis indicated that the GH3-like motif in Ibm1 has an important supportive role in IAP-antagonist function and can trigger cell death under certain conditions.     

Model-based analysis of rapid event-related functional near-infrared spectroscopy (NIRS) data: a parametric validation study

To validate the usefulness of a model-based analysis approach according to the general linear model (GLM) for functional near-infrared spectroscopy (fNIRS) data, a rapid event-related paradigm with an unpredictable stimulus sequence was applied to 15 healthy subjects. A parametric design was chosen wherein four differently graded contrasts of a flickering checkerboard were presented, allowing directed hypotheses about the rank order of the evoked hemodynamic response amplitudes. The results indicate the validity of amplitude estimation by three main findings (a) the GLM approach for fNIRS data is capable to identify human brain activation in the visual cortex with inter-stimulus intervals of 4-9 s (6.5 s average) whereas in non-visual areas no systematic activation was detectable; (b) the different contrast level intensities lead to the hypothesized rank order of the GLM amplitude parameters: visual cortex activation evoked by highest contrast>moderate contrast>lowest contrast>no stimulation; (c) analysis of null-events (no stimulation) did not produce any significant activation in the visual cortex or in other brain areas. We conclude that a model-based GLM approach delivers valid fNIRS amplitude estimations and enables the analysis of rapid event-related fNIRS data series, which is highly relevant in particular for cognitive fNIRS studies.     

Boolarra virus: a member of the Nodaviridae isolated from Oncopera intricoides (Lepidoptera: Hepialidae)

A small RNA virus with a bipartite genome was isolated from Oncopera intricoides. It was named Boolarra virus (BoV). The particle had a diameter of 30 nm, a sedimentation coefficient of 140S, and a buoyant density of 1.34 g/ml in CsCl. The capsid proteins were examined by SDS-PAGE and consisted of a major species of molecular weight (mol. wt.) 38,000 and a minor one of mol. wt. 40,600. The particle contained 21.2% RNA which was divided between a 22S and a 17S species whose mol. wts. were 1.03 x 10(6) and 0.47 x 10(6), respectively. These properties were comparable to those of the Nodaviridae and established BoV as a member of the family. Serologically, BoV shared antigenic determinants with both Nodamura virus and black beetle virus.     

Discrete-trial functional analysis (DTFA) approach appears practical and effective

This review provides a summary and appraisal commentary on the treatment review by Chezan, L. C., Drasgow, E., & Martin, C. A. (2014). Discrete-trial functional analysis and functional communication training with three adults with intellectual disabilities and problem behavior. Journal of Behavioral Education, 23, 221–246.

Source of funding and declaration of interests. There were no funding sources acknowledged and no interests declared.     

Identification and functional analysis of a novel chorion protein essential for egg maturation in the brown planthopper

In insect eggs, the chorion has the essential function of protecting the embryo from external agents during development while allowing gas exchange for respiration. In this study, we found a novel gene, Nilaparvata lugens chorion protein (NlChP), that is involved in chorion formation in the brown planthopper, Nilaparvata lugens. NlChP was highly expressed in the follicular cells of female adult brown planthoppers. Knockdown of NlChP resulted in oocyte malformation and the inability to perform oviposition, and electron microscopy showed that the malformed oocytes had thin and rough endochorion layers compared to the control group. Liquid chromatography with tandem mass spectrometry analysis of the eggshell components revealed four unique peptides that were matched to NlChP. Our results demonstrate that NlChP is a novel chorion protein essential for egg maturation in N. lugens, a hemipteran insect with telotrophic meroistic ovaries. NlChP may be a potential target in RNA interference‐based insect pest management.     

Early determination of fetal sex using transvaginal sonography: technique and pitfalls

Transvaginal sonography (TVS) enables sex determination at an early stage of pregnancy. The morphologic features of fetal external genitalia at 13 weeks to 16 weeks, menstrual age, are different from those seen later in pregnancy; therefore an attempt to determine fetal gender at this early stage by the same criteria as those used later is hazardous, especially for determining the male sex. The main diagnostic criteria for male gender determination by TVS are the "dome" sign representing the sonographic visualization of the fetal scrotum, the cranially directed phallus, and the longitudinal raphe at the base of the penis. The diagnostic criteria for female gender are the 2 or 4 parallel lines representing the labial folds and the caudally directed phallus (clitoris). The length of the fetal phallus at this early stage is not diagnostic and may be the main pitfall to the unexperienced sonographer. Between weeks 13 and 14 sex diagnosis was possible in 130/171 pregnancies (76%) in our first 2 years and 188/235 (80%) in our last 2 years of experience. Between weeks 15 and 16 sex diagnosis was possible in 122/139 pregnancies (88%) during our first 2 years and 96.7% (528/546) during the last 2 years of experience. The accuracy rate for fetal male gender identification increased from 91.7% during the first 2 years of TVS experience to 99.7% during the last 2 years of TVS experience, and the accuracy rate for female gender identification, increased from 93.3% to 100%, respectively, applying the above criteria and based on acquired experience of early fetal sex identification by TVS early in gestation. Early and precise determination of fetal sex is possible and might avoid invasive procedures such as amniocentesis.     

Phenotypic and functional analysis of thymic nurse cell (TNC)-lymphocytes

Thymic nurse cells (TNC) are epithelial cells containing intact T-cells engulfed within membrane-lined vacuoles in their cytoplasm. Since thymic epithelial cells strongly express MHC class I and II antigens, these lymphoid-epithelial complexes have been considered to provide an optimal micro-environment for the process of self-recognition and/or clonal deactivation of thymocytes. The present experiments were designed to further analyze the phenotypical stage of differentiation and the functional properties of intra-TNC lymphocytes (TNC-L) at a single cell level using the chorionallantoic membrane (CAM) assay, which can only be performed in avian species and, thus, presents a unique possibility to circumvent technical problems for the functional study of TNC-L in mammals. In immunofluorescence analyses with monoclonal antibodies (mAb) to the chicken alpha/beta and gamma/delta T cell receptors (TCR) and the CD3, CD4, and CD8 equivalents, an enrichment was found of CD3+/CD4+/CD8-, CD3+/CD4-/CD8+, TCR alpha/beta + and TCR gamma/delta + cells inside TNC, as compared with extra-TNC thymocytes. Assessing micromanipulated, single chicken TNC in the CAM assay, we also demonstrated at a clonal level that TNC-L possess a strong graft-versus-host (GvH) reactivity in allogeneic combinations. This reaction showed morphological, phenotypical and functional characteristics of a classical graft-versus-host reaction (GvHR). The efficiency to induce a GvHR was higher for TNC-L than peripheral blood lymphocytes (PBL) or thymocytes from the same donor. Surprisingly, in syngeneic combinations TNC-L also react against self-MHC molecules with high frequency. The frequency in syngeneic combinations is, however, lower than in allogeneic combinations.(ABSTRACT TRUNCATED AT 250 WORDS)